Methods of promoting antitumor immunity by administering CD40 agonists and alpha-galactosyl ceramide

ABSTRACT

Adjuvant combinations comprising at least one NKT activator, such as alpha-galactosylceramide (α-Gal-Cer) or iGb3, a CD40 agonist and optionally an antigen are disclosed. The use of these immune adjuvants for treatment of various chronic diseases such as cancers is also provided.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. 371 toPCT/US2008/005341 filed on Apr. 25, 2008, which claims priority to U.S.Provisional Application No. 60/907,976 filed on Apr. 25, 2007, all whichare incorporated by reference in their entirety herein.

FIELD OF THE INVENTION

The invention generally relates to synergistic adjuvant combinationswhich upregulate CD70 expression on immune cells and promote antigenspecific cellular immunity. More particularly, the invention relates toa specific synergistic combination comprising a Natural Killer T cell(NKT) activator, e.g., alpha-galactosylceramide (α-Gal-Cer) or iGb3, anda CD40 agonist, e.g., an agonistic anti-CD40 antibody or a CD40Lpolypeptide or conjugate, and optionally further including a targetantigen. Still further the invention provides novel immune therapiescomprising the administration of such synergistic adjuvant combinationsor protein conjugates to enhance antigen specific cellular immunity,i.e., CD8+ immunity. Specifically, the use of compositions comprisingthese adjuvant combinations and/or or polypeptide conjugates as immuneadjuvants for treating various chronic diseases including cancer,infectious diseases, autoimmune diseases, allergic and inflammatorydiseases is also taught.

BACKGROUND OF THE INVENTION

The body's defense system against microbes as well as the body's defenseagainst other chronic diseases such as those affecting cellproliferation is mediated by early reactions of the innate immune systemand by later responses of the adaptive immune system. Innate immunityinvolves mechanisms that recognize structures which are for examplecharacteristic of the microbial pathogens and that are not present onmammalian cells. Examples of such structures include bacterialliposaccharides (LPS), viral double stranded DNA, and unmethylated CpGDNA nucleotides. The effector cells of the innate immune response systemcomprise neutrophils, macrophages, and natural killer cells (NK cells).In addition to innate immunity, vertebrates, including mammals, haveevolved immunological defense systems that are stimulated by exposure toinfectious agents and that increase in magnitude and effectiveness witheach successive exposure to a particular antigen. Due to its capacity toadapt to a specific infection or antigenic insult, this immune defensemechanism has been described as adaptive immunity. There are two typesof adaptive immune responses, called humoral immunity, involvingantibodies produced by B lymphocytes, and cell-mediated immunity,mediated by T lymphocytes.

Two types of major T lymphocytes have been described, CD8+ cytotoxiclymphocytes (CTLs) and CD4 helper cells (Th cells). CD8+ T cells areeffector cells that, via the T cell receptor (TCR), recognize foreignantigens presented by class I MHC molecules on, for instance, virally orbacterially infected cells. Upon recognition of foreign antigens, CD8+cells undergo an activation, maturation and proliferation process. Thisdifferentiation process results in CTL clones which have the capacity ofdestroying the target cells displaying foreign antigens. T helper cellson the other hand are involved in both humoral and cell-mediated formsof effector immune responses. With respect to the humoral, or antibodyimmune response, antibodies are produced by B lymphocytes throughinteractions with Th cells. Specifically, extracellular antigens, suchas circulating microbes, are taken up by specialized antigen-presentingcells (APCs), processed, and presented in association with class IImajor histocompatibility complex (MHC) molecules to CD4+ Th cells. TheseTh cells in turn activate B lymphocytes, resulting in antibodyproduction. The cell-mediated, or cellular, immune response, incontrast, functions to neutralize microbes which inhabit intracellularlocations, such as after successful infection of a target cell. Foreignantigens, such as for example, microbial antigens, are synthesizedwithin infected cells and resented on the surfaces of such cells inassociation with Class I MHC molecules. Presentation of such epitopesleads to the above-described stimulation of CD8+ CTLs, a process whichin turn is also stimulated by CD4+ Th cells. Th cells are composed of atleast two distinct subpopulations, termed Th1 and Th2 cells. The Th1 andTh2 subtypes represent polarized populations of Th cells whichdifferentiate from common precursors after exposure to antigen.

Each T helper cell subtype secretes cytokines that promote distinctimmunological effects that are opposed to one another and thatcross-regulate each other's expansion and function. Th1 cells secretehigh amounts of cytokines such as interferon (IFN) gamma, tumor necrosisfactor-alpha (TNF-alpha), interleukin-2 (IL-2), and IL-12, and lowamounts of IL-4. Th1 associated cytokines promote CD8+ cytotoxic Tlymphocyte T lymphocyte (CTL) activity and are most frequentlyassociated with cell-mediated immune responses against intracellularpathogens. In contrast, Th2 cells secrete high amounts of cytokines suchas IL-4, IL-13, and IL-10, but low IFN-gamma, and promote antibodyresponses. Th2 responses are particularly relevant for humoralresponses, such as protection from anthrax and for the elimination ofhelminthic infections.

Whether a resulting immune response is Th1 or Th2-driven largely dependson the pathogen involved and on factors in the cellular environment,such as cytokines. Failure to activate a T helper response, or thecorrect T helper subset, can result not only in the inability to mount asufficient response to combat a particular pathogen, but also in thegeneration of poor immunity against reinfection. Many infectious agentsare intracellular pathogens in which cell-mediated responses, asexemplified by Th1 immunity, would be expected to play an important rolein protection and/or therapy. Moreover, for many of these infections ithas been shown that the induction of inappropriate Th2 responsesnegatively affects disease outcome. Examples include M tuberculosis, S.mansoni, and also counterproductive Th2-like dominated immune responses.Lepromatous leprosy also appears to feature a prevalent, butinappropriate, Th2-like response. HIV infection represents anotherexample. There, it has been suggested that a drop in the ratio ofTh1-like cells to other Th cell populations can play a critical role inthe progression toward disease symptoms.

As a protective measure against infectious agents, vaccination protocolsfor protection from some microbes have been developed. Vaccinationprotocols against infectious pathogens are often hampered by poorvaccine immunogenicity, an inappropriate type of response (antibodyversus cell-mediated immunity), a lack of ability to elicit long-termimmunological memory, and/or failure to generate immunity againstdifferent serotypes of a given pathogen. Current vaccination strategiestarget the elicitation of antibodies specific for a given serotype andfor many common pathogens, for example, viral serotypes or pathogens.Efforts must be made on a recurring basis to monitor which serotypes areprevalent around the world. An example of this is the annual monitoringof emerging influenza A serotypes that are anticipated to be the majorinfectious strains.

To support vaccination protocols, adjuvants that would support thegeneration of immune responses against specific infectious diseasesfurther have been developed. For example, aluminum salts have been usedas a relatively safe and effective vaccine adjuvants to enhance antibodyresponses to certain pathogens. One of the disadvantages of suchadjuvants is that they are relatively ineffective at stimulating acell-mediated immune response and produce an immune response that islargely Th2 biased.

It is now widely recognized that the generation of protective immunitydepends not only on exposure to antigen, but also the context in whichthe antigen is encountered. Numerous examples exist in whichintroduction of a novel antigen into a host in a non-inflammatorycontext generates immunological tolerance rather than long-term immunitywhereas exposure to antigen in the presence of an inflammatory agent(adjuvant) induces immunity. (Mondino et al., Proc. Natl. Acad. Sci.,USA 93:2245 (1996); Pulendran et al., J. Exp. Med. 188:2075 (1998);Jenkins et al., Immunity 1:443 (1994); and Kearney et al., Immunity1:327 (1994)).

A naturally occurring molecule known to regulate adaptive immunity isCD40. CD40 is a member of the TNF receptor superfamily and is essentialfor a spectrum of cell-mediated immune responses and required for thedevelopment of T cell dependent humoral immunity (Aruffo et al., Cell72:291 (1993); Farrington et al., Proc Natl Acad. Sci., USA 91:1099(1994); Renshaw et al., J Exp Med 180:1889 (1994)). In its natural role,CD40-ligand expressed on CD4+ T cells interacts with CD40 expressed onDCs or B cells, promoting increased activation of the APC and,concomitantly, further activation of the T cell (Liu et al Semin Immunol9:235 (1994); Bishop et al., Cytokine Growth Factor Rev 14:297 (2003)).For DCs, CD40 ligation classically leads to a response similar tostimulation through TLRs such as activation marker upregulation andinflammatory cytokine production (Quezada et al. Annu Rev Immunol 22:307(2004); O'Sullivan B and Thomas R Crit. Rev Immunol 22:83 (2003)). Itsimportance in CD8 responses was demonstrated by studies showing thatstimulation of APCs through CD40 rescued CD4-dependent CD8+ T cellresponses in the absence of CD4 cells (Lefrancois et al., J. Immunol.164:725 (2000); Bennett et al., Nature 393:478 (1998); Ridge et al.,Nature 393:474 (1998); Schoenberger et al., Nature 393:474 (1998). Thisfinding sparked much speculation that CD40 agonists alone couldpotentially rescue failing CD8+ T cell responses in some diseasesettings.

Other studies, however, have demonstrated that CD40 stimulation aloneinsufficiently promotes long-term immunity. In some model systems,anti-CD40 treatment alone insufficiently promoted long-term immunity. Insome model systems, anti-CD40 treatment alone can result in ineffectiveinflammatory cytokine production, the deletion of antigen-specific Tcells (Mauri et al. Nat Med 6:673 (2001); Kedl et al. Proc Natl Acad.Sci., USA 98:10811 (2001)) and termination of B cell responses (Ericksonet al., J Clin Invest 109:613 (2002)). Also, soluble trimerized CD40ligand has been used n the clinic as an agonist for the CD40 pathway andwhat little has been reported is consistent with the conclusion thatstimulation of CD40 alone fails to reconstitute all necessary signalsfor long term CD8+ T cell immunity (Vonderheide et al., J Clin Oncol19:3280 (2001)).

Various agonistic antibodies have been reported by different groups. Forexample, one mAb CD40.4 (5c3) (PharMingen, San Diego Calif.) has beenreported to increase the activation between CD40 and CD40L byapproximately 30-40%. (Schlossman et al., Leukocyte Typing, 1995,1:547-556). Also, Seattle Genetics in U.S. Pat. No. 6,843,989 allege toprovide methods of treating cancer in humans using an agonisticanti-human CD40 antibody. Their antibody is purported to deliver astimulatory signal, which enhances the interaction of CD40 and CD40L byat least 45% and enhances CD40L-mediated stimulation and possess in vivoneoplastic activity. They obtain this antibody from S2C6, an agonisticanti-human CD40 antibody previously shown to deliver stronggrowth-promoting signals to B lymphocytes. (Paulie et al., 1989, J.Immunol. 142:590-595).

Because of the activity of CD40 in innate and adaptive immune responses,various CD40 agonists have been explored for usage as vaccine adjuvantsand in therapies in wherein enhanced cellular immunity is desired.Recently, it was demonstrated that immunization with antigen incombination with some TLR agonists and anti-CD40 treatment (combinedTLR/CD40 agonist immunization) induces potent CD8+ T cell expansion,eliciting a response 10-20 fold higher than immunization with eitheragonist alone (Ahonen et al., J Exp Med 199:775 (2004)). This was thefirst demonstration that potent CD8+ T cell responses can be generatedin the absence of infection with a viral or microbial agent. Antigenspecific CD8+ T cells elicited by combined TLR/CD40 agonist immunizationdemonstrate lytic function, gamma interferon production, and enhancedsecondary responses to antigenic challenge. Synergistic activity withanti-CD40 in the induction of CD8+ T cell expansion has been shown withagonists of TLR1/6, 2/6, 3, 4, 5, 7 and 9. This suggests that combinedTLR/CD40 agonist immunization can reconstitute all of the signalsrequired to elicit profound acquired cell-mediated immunity.

It is known that NKT cells are immunoregulatory T-lymphocytes thatexpress a T cell receptor which is restricted by the non-polymorphicCD1d antigen presenting molecule. NKT cells recognize the CD1d-presentedglycolipid, alpha-galactosylceramide (α-Gal-Cer). Upon recognition ofα-Gal-Cer, NKT cells become activated and produce cytokines includingIL-4, IL-10, IL-13 and IFN-γ, and as such, they can either upregulate ordownregulate immune responses by promoting the secretion of immuneregulatory cytokines. Mice which are devoid of NKT cells are moresusceptible to bacterial infections and resistance to some tumors,demonstrating an important role for NKT cells in host defense. It hasalso been shown that activation of NKT cells by the administration ofα-Gal-Cer, as a monotherapy, to mice can enhance the immunity to tumorswith limited success (Matsuyoshi, H., S. Hirata, Y. Yoshitake, Y.Motomura, D. Fukuma, A. Kurisaki, T. Nakatsura, Y. Nishimura, and S.Senju. 2005. Therapeutic effect of alpha-galactosylceramide-loadeddendritic cells genetically engineered to express SLC/CCL21 along withtumor antigen against peritoneally disseminated tumor cells. Cancer Sci.96:889-896; Crowe, N. Y., J. M. Coquet, S. P. Berzins, K. Kyparissoudis,R. Keating, D. G. Pellicci, Y. Hayakawa, D. I. Godfrey, and M. J. Smyth.2005. Differential antitumor immunity mediated by NKT cell subsets invivo. J. Exp. Med. 202:1279-1288; Smyth, M. J., N. Y. Crowe, Y.Hayakawa, K. Takeda, H. Yagita, and D. I. Godfrey. 2002. NKTcells—conductors of tumor immunity? Curr. Opin. Immunol. 14:165-171).

To increase the effectiveness of an adaptive immune response, such as ina vaccination protocol or during a microbial infection, it is thereforeimportant to develop novel, more effective, vaccine adjuvants. Thepresent invention satisfies this need and provides other advantages aswell. Previously, the present inventors have reported novel synergisticadjuvants comprising the combination of a toll like receptor (TLR)agonist and a CD40 agonist. These agonists in combination elicit asynergistic effect on cellular immunity. These synergistic adjuvants andthe prophylactic and therapeutic applications thereof are disclosed inUS published patent application US20040141950 published on Jul. 22,2004, and which patent application is incorporated by reference in itsentirety herein. This invention relates to the discovery of anothersynergistic adjuvant combination and the use thereof as a therapeutic orprophylactic immune potentiating combination.

SUMMARY OF THE INVENTION

This invention relates to synergistic immune adjuvants comprising thecombination of (i) at least one NKT activator, e.g.,alpha-galactosylceramide (α-GalCer) or iGb3; and (ii) at least one CD40agonist such as a CD40 agonistic antibody or fragment thereof or a CD40Lpolypeptide such as a monomeric or multimeric (trimeric) CD40L protein,CD40L protein fragment, or conjugate containing CD40L and (iii)optionally an antigen against which a cellular immune response isdesirably elicited. The present invention further relates to the use ofsuch combinations as immune adjuvants and for treating conditionswherein T cell immunity is desirably enhanced.

As described in detail infra, these immune combinations may beadministered to a host in need of such treatment as a means of:

-   -   (i) inducing higher frequencies of antigen-specific CD8+ T cells        relative to the administration of only a CD40 agonist or an NKT        activator;    -   (ii) inducing higher quantities of antigen-specific CD8+ T cells        relative to the administration of only a CD40 agonist or an NKT        activator; and    -   (iii) enhancing tumor immunity relative to the administration of        only a CD40 agonist or an NKT activator.

These immune adjuvant combinations which optionally may further includean antigen may be used in treating any disease or condition wherein theabove-identified enhanced cellular immune responses are therapeuticallydesirable, especially infectious diseases, proliferative disorders suchas cancer, allergy, autoimmune disorders, inflammatory disorders, andother chronic diseases wherein enhanced cellular immunity is a desiredtherapeutic outcome. Preferred applications of the invention includeespecially the treatment of a chronic disease such as cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 contains an experiment showing the effect of combinedadministration of anti-CD40 and α-GalCer and that such administrationsynergistically enhances the expansion of antigen-specific T cells invivo.

FIG. 2 contains an experiment that quantifies immune responses as aresult of immunization with combined anti-CD40 and α-GalCer.

FIG. 3 contains an experiment in mice immunized with melanoma cellsshowing that CD40/TLR* vaccination therapeutically intervenes insystemic melanoma.

FIG. 4 contains an experiment showing that anti-CD40 and α-GalCeradministration induces CD70 expression on DCs in vivo. (In thisexperiment mice were injected with combinations of the following: 2 ugα-GalCer, 50 ug polyIC, and/or 50 ug anti-CD40 i.p. Spleens were takenat 18-20 hours post injection).

FIG. 5 contains an experiment showing that α-GalCer/CD40 immunizationelicits a potent CD8+ T cell response that is STAT4, IFNγ and IFNabindependent but is STAT1 and T-bet independent.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of a synergistic immuneadjuvant combination comprising synergistically effective amounts of (i)at least one natural killer-T cell (NKT) activating agent such asalpha-galactosylceramide (α-GalCer) or another NKT cell activatingagent; (ii) at least one CD40 agonist and (ii) optionally at least oneantigen. These adjuvants may be used to (i) expand antigen-specificeffector CD8 T cells, and/or (ii) to enhance immune responses. Basedthereon these synergistic adjuvants can be used prophylactically andtherapeutically to treat subjects having conditions wherein enhancedcellular immunity is desired such as cancer and other proliferativedisorders, viral and other infections, autoimmunity, inflammatorydiseases and allergic disorders. Particularly, the subject synergisticadjuvants may be used vaccine adjuvant. The basic components of such avaccine adjuvant would therefore be an NKT cell activator, a CD40agonist and the addition of a tumor or infectious disease antigen, ifwarranted. Such an invention would be useful in any situation where onewould like to enhance immune responses, like cancer or infectiousdisease, or modify immune responses like in autoimmunity.

More specifically, the present invention provides a novel synergisticagonistic combination comprising an NKT activator, e.g.,alpha-galactosylceramide (α-Gal-Cer) or iGb3 and a CD40 agonist (forexample a CD40L protein or fragment or derivative or multimeric thereofor an agonistic antibody or antibody fragment that binds CD40,preferably human CD40) and optionally an antigen. These adjuvantcombinations when administered to a host, preferably a human, may beused to generate enhanced antigen specific cellular immune responses.

In preferred embodiments the NKT activator is alpha-galactosylceramide(α-Gal-Cer), isoglobotrihexosylceramide (iGb3) or any other moleculeswhich can stimulate NKT such as, for example, other glycolipidspresented by CD1d antigen-presenting molecules.

Also, the invention provides methods of using said synergistic adjuvantcombinations and vehicles containing to a host in which an antigenspecific immune response is desirably elicited, for example a personwith a chronic disease such as cancer or an infectious or allergicdisorder producing said composition.

Still further the invention provides compositions comprising said novelsynergistic NKT activator/CD40 agonist combinations which are suitablefor administration to a host in order to elicit an enhancedantigen-specific cellular immune response.

Particularly, the invention provides novel methods of immunotherapycomprising the administration of said novel synergistic adjuvantcombination to a host in need of such treatment in order to elicit anenhanced antigen specific cellular immune response. In preferredembodiments these compositions and conjugates will be administered to asubject with or at risk of developing a cancer, an infection,particularly a chronic infectious diseases e.g., involving a virus,bacteria or parasite; or an autoimmune, inflammatory or allergiccondition. Particularly, in a preferred embodiment, the invention may beused to elicit antigen specific cellular immune responses againstmelanoma.

Thus, this invention provides for the development of potent vaccinesagainst cancer and other proliferative diseases such as autoimmunediseases, allergic disorders, and inflammatory diseases as well aschronic infectious diseases involving viruses, bacteria, fungi orparasites, where effective treatment requires the quantity and qualityof cellular immunity that combined NKT activator/CD40 agonistimmunization is capable of generating.

APPLICATIONS OF THE INVENTION

The invention provides novel adjuvant combinations comprising at leastone NKT activator, at least one CD40 agonist and optionally an antigen.The invention embraces the use of any antigen in combination with thesubject NKT activators and CD40 agonists against which an enhancedcellular immune response is therapeutically desirable. In someembodiments the antigen may be administered separate from the NKTactivator, or the host may be naturally exposed to the antigen. In someembodiments all three moieties, i.e., the CD40 agonist such as anti-CD40antibody, the NKT activator and the antigen, may be administeredconcurrently. Additionally, in some embodiments all three moieties,i.e., the CD40 agonist such as anti-CD40 antibody, the NKT activator andthe antigen, may be co-administered as separate discrete entities.Preferably all these moieties are administered substantiallyconcurrently in order to achieve the desired synergistic enhancement incellular immunity. These moieties may be administered in any order.

Exemplary antigens include but are not limited to bacterial, viral,parasitic, allergens, autoantigens and tumor associated antigens.Particularly, the antigen can include protein antigens, peptides, wholeinactivated organisms, and the like.

Specific examples of antigens that can be used in the invention includeantigens from hepatitis A, B, C or D, influenza virus, Listeria,Clostridium botulinum, tuberculosis, tularemia, Variola major(smallpox), viral hemorrhagic fevers, Yersinia pestis (plague), HIV,herpes, papilloma virus, and other antigens associated with infectiousagents. Other antigens include antigens associated with a tumor cell,antigens associated with autoimmune conditions, allergy and asthma.Administration of such an antigen in conjunction with the subjectagonist combination flagellin and an anti-CD40 antibody can be used in atherapeutic or prophylactic vaccine for conferring immunity against suchdisease conditions.

In some embodiments the methods and compositions can be used to treat anindividual at risk of having an infection or has an infection byincluding an antigen from the infectious agent. An infection refers to adisease or condition attributable to the presence in the host of aforeign organism or an agent which reproduce within the host. A subjectat risk of having an infection is a subject that is predisposed todevelop an infection. Such an individual can include for example asubject with a known or suspected exposure to an infectious organism oragent. A subject at risk of having an infection can also include asubject with a condition associated with impaired ability to mount animmune response to an infectious agent or organism, for example asubject with a congenital or acquired immunodeficiency, a subjectundergoing radiation or chemotherapy, a subject with a burn injury, asubject with a traumatic injury, a subject undergoing surgery, or otherinvasive medical or dental procedure, or similarly immunocompromisedindividual.

Infections which may be treated or prevented with the vaccinecompositions of this invention include bacterial, viral, fungal, andparasitic. Other less common types of infection also include arerickettsiae, mycoplasms, and agents causing scrapie, bovine spongiformencephalopathy (BSE), and prion diseases (for example kuru andCreutzfeldt-Jacob disease). Examples of bacteria, viruses, fungi, andparasites that infect humans are well know. An infection may be acute,subacute, chronic or latent and it may be localized or systemic.Furthermore, the infection can be predominantly intracellular orextracellular during at least one phase of the infectious organism'sagent's life cycle in the host.

Bacteria infections against which the subject vaccines and methods maybe used include both Gram negative and Gram positive bacteria. Examplesof Gram positive bacteria include but are not limited to Pasteurellaspecies, Staphylococci species, and Streptococci species. Examples ofGram negative bacteria include but are not limited to Escherichia coli,Pseudomonas species, and Salmonella species. Specific examples ofinfectious bacteria include but are not limited to Heliobacter pyloris,Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria spp. (forexample M. tuberculosis, M. avium, M. intracellilare, M. kansaii, M.gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseriameningitidis, Listeria monocytogeners, Streptococcus pyogenes, (group AStreptococcus), Streptococcus agalactiae(Group B Streptococcus),Streptococcus (viridans group), Streptococcus faecalis, streptococcusbovis, Streptococcus (anaerobic spp.), Streptococcus pneumoniae,pathogenic Campylobacter spp., Enterococcus spp., Haemophilusinfluenzae, Bacillus anthracis, Corynebacterium diptheriae,Corynebacterium spp., Erysipelothrix rhusiopathie, Clostridiumperfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasteurella multocida, Bacteroides spp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelii.

Examples of viruses that cause infections in humans include but are notlimited to Retroviridae (for example human deficiency viruses, such asHIV-1 (also referred to as HTLV-III), HIV-II, LAC or IDLV-III/LAV orHIV-III and other isolates such as HIV-LP), Picornaviridae (for examplepoliovirus, hepatitis A, enteroviruses, human Coxsackie viruses,rhinoviruses, echoviruses), Calciviridae (for example strains that causegastroenteritis), Togaviridae (for example equine encephalitis viruses,rubella viruses), Flaviviridae (for example dengue viruses, encephalitisviruses, yellow fever viruses) Coronaviridae (for examplecoronaviruses), Rhabdoviridae (for example vesicular stomata viruses,rabies viruses), Filoviridae (for example Ebola viruses) Paramyxoviridae(for example parainfluenza viruses, mumps viruses, measles virus,respiratory syncytial virus), Orthomyxoviridae (for example influenzaviruses), Bungaviridae (for example Hataan viruses, bunga viruses,phleoboviruses, and Nairo viruses), Arena viridae (hemorrhagic feverviruses), Reoviridae (for example reoviruses, orbiviruses, rotaviruses),Bimaviridae, Hepadnaviridae (hepatitis B virus), Parvoviridae(parvoviruses), Papovaviridae (papilloma viruses, polyoma viruses),Adenoviridae (adenoviruses), Herpeviridae (for example herpes simplexvirus (HSV) I and II, varicella zoster virus, pox viruses) andIridoviridae (for example African swine fever virus) and unclassifiedviruses (for example the etiologic agents of Spongiformencephalopathies, the agent of delta hepatitis, the agents of non-A,non-B hepatitis (class 1 enterally transmitted; class 2 parenterallytransmitted such as Hepatitis C); Norwalk and related viruses andastroviruses.

Examples of fungi include Aspergillus spp., Coccidoides immitis,Cryptococcus neoformans, Candida albicans and other Candida spp.,Blastomyces dermatidis, Histoplasma capsulatum, Chlamydia trachomatis,Nocardia spp., and Pneumocytis carinii.

Parasites include but are not limited to blood-borne and/or tissueparasites such as Babesia microti, Babesi divergans, Entomoebahistolytica, Giarda lamblia, Leishmania tropica, Leishmania spp.,Leishmania braziliensis, Leishmania donovdni, Plasmodium falciparum,Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Toxoplasmagondii, Trypanosoma gambiense and Trypanosoma rhodesiense (Africansleeping sickness), Trypanosoma cruzi (Chagus' disease) and Toxoplasmagondii, flat worms, and round worms.

As noted this invention further embraces the use of the subjectconjugates in treating proliferative diseases such as cancers, and, in apreferred embodiment, in treating melanoma. Cancer is a condition ofuncontrolled growth of cells which interferes with the normalfunctioning of bodily organs and systems. A subject that has a cancer isa subject having objectively measurable cancer cells present in thesubject's body. A subject at risk of developing cancer is a subjectpredisposed to develop a cancer, for example based on family history,genetic predisposition, subject exposed to radiation or othercancer-causing agent. Cancers which migrate from their original locationand seed vital organs can eventually lead to the death of the subjectthrough the functional deterioration of the affected organ.Hematopoietic cancers, such as leukemia, are able to out-compete thenormal hematopoietic compartments in a subject thereby leading tohematopoietic failure (in the form of anemia, thrombocytopenia andneutropenia), ultimately causing death.

A metastasis is a region of cancer cells, distinct from the primarytumor location, resulting from the dissemination of cancer cells fromthe primary tumor to other parts of the body. At the time of diagnosisof the primary tumor mass, the subject may be monitored for the presenceof metastases. Metastases are often detected through the sole orcombined use of magnetic resonance imaging (MRI), computed tomography(CT), scans, blood and platelet counts, liver function studies, chest-X-rays and bone scans in addition to the monitoring of specificsymptoms.

The adjuvant combinations and compositions containing according to theinvention can be used to treat a variety of cancers or subjects at riskof developing cancer, by the inclusion of a tumor-associated-antigen(TAA), or DNA encoding. This is an antigen expressed in a tumor cell.Examples of such cancers include breast, prostate, colon, blood cancerssuch as leukemia, chronic lymphocytic leukemia, and the like. Thevaccination methods of the invention can be used to stimulate an immuneresponse to treat a tumor by inhibiting or slowing the growth of thetumor or decreasing the size of the tumor. A tumor associated antigencan also be an antigen expressed predominantly by tumor cells but notexclusively.

Additional cancers include but are not limited to basal cell carcinoma,biliary tract cancer, bladder cancer, bone cancer, brain and centralnervous system (CNS) cancer, cervical cancer, choriocarcinoma,colorectal cancers, connective tissue cancer, cancer of the digestivesystem, endometrial cancer, esophageal cancer, eye cancer, head and neckcancer, gastric cancer, intraepithelial neoplasm, kidney cancer, larynxCancer, liver cancer, lung cancer (small cell, large cell), lymphomaincluding Hodgkin's lymphoma and non-Hodgkin's lymphoma; neuroblastoma;oral cavity cancer (for example lip, tongue, mouth and pharynx); ovariancancer; pancreatic cancer; retinoblastoma; rhabdomyosarcoma; rectalcancer; cancer of the respiratory system; sarcoma; skin cancer; stomachcancer; testicular cancer; thyroid cancer; uterine cancer; cancer of theurinary system; as well as other carcinomas and sarcomas.

The adjuvant combinations and compositions containing according to theinvention can also be used to treat autoimmune diseases such as multiplesclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis or otherautoimmune disorders. Other autoimmune disease which potentially may betreated with the vaccines and immune adjuvants of the invention includeCrohn's disease and other inflammatory bowel diseases such as ulcerativecolitis, systemic lupus erythematosus (SLE), autoimmuneencephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis,Goodpasture's syndrome, pemphigus, Graves disease, autoimmune hemolyticanemia, autoimmune thrombocytopenic purpura, scleroderma withanti-collagen antibodies, mixed connective tissue disease, polymyositis,pernicious anemia, idiopathic Addison's disease, autoimmune associatedinfertility, glomerulonephritis (for example crescenticglomerulonephritis, proliferative glomerulonephritis), bullouspemphigoid, Sjogren's syndrome, psoriatic arthritis, insulin resistance,autoimmune diabetes mellitus (type 1 diabetes mellitus; insulindependent diabetes mellitus), autoimmune hepatitis, autoimmunehemophilia, autoimmune lymphoproliferative syndrome (ALPS), autoimmunehepatitis, autoimmune hemophilia, autoimmune lymphoproliferativesyndrome, autoimmune uveoretinitis, and Guillain-Bare syndrome.Recently, arteriosclerosis and Alzheimer's disease have been recognizedas autoimmune diseases. Thus, in this embodiment of the invention theantigen will be a self-antigen against which the host elicits anunwanted immune response that contributes to tissue destruction and thedamage of normal tissues.

The adjuvant combinations and compositions containing according to theinvention can also be used to treat asthma and allergic and inflammatorydiseases. Asthma is a disorder of the respiratory system characterizedby inflammation and narrowing of the airways and increased reactivity ofthe airways to inhaled agents. Asthma is frequently although notexclusively associated with atopic or allergic symptoms. Allergy isacquired hypersensitivity to a substance (allergen). Allergic conditionsinclude eczema, allergic rhinitis, or coryza, hay fever, bronchialasthma, urticaria, and food allergies and other atopic conditions. Anallergen is a substance that can induce an allergic or asthmaticresponse in a susceptible subject. There are numerous allergensincluding pollens, insect venoms, animal dander, dust, fungal spores,and drugs.

Examples of natural and plant allergens include proteins specific to thefollowing genera: Canine, Dermatophagoides, Felis, Ambrosia, Lotium,Cryptomeria, Alternaria, Alder, Alinus, Betula, Quercus, Olea,Artemisia, Plantago, Parietaria, Blatella, Apis, Cupressus, Juniperus,Thuya, Chamaecyparis, Periplanet, Agopyron, Secale, Triticum, Dactylis,Festuca, Poa, Avena, Holcus, Anthoxanthum, Arrhenatherum, Agrostis,Phleum, Phalaris, Paspalum, Sorghum, and Bromis.

It is understood that the adjuvant combinations and compositionscontaining according to the invention can be combined with othertherapies for treating the specific condition, e.g., infectious disease,cancer or autoimmune condition. For example in the case of cancer theinventive methods may be combined with chemotherapy or radiotherapy.

Methods of making compositions as vaccines are well known to thoseskilled in the art. The effective amounts of the NKT activator, CD40agonist and optionally an antigen can be determined empirically, but canbe based on immunologically effective amounts in animal models. Factorsto be considered include the antigenicity, the formulation, the route ofadministration, the number of immunizing doses to be administered, thephysical condition, weight, and age of the individual, and the like.Such factors are well known to those skilled in the art and can bedetermined by those skilled in the art (see for example Paoletti andMcInnes, eds., Vaccines, from Concept to Clinic: A Guide to theDevelopment and Clinical Testing of Vaccines for Human Use CRC Press(1999)). As disclosed herein it is understood that the subject DNAs orprotein conjugates can be administered alone or in conjunction withother adjuvants.

The adjuvants of the invention can be administered locally orsystemically by any method known in the art including but not limited tointramuscular, intravenous, intradermal, subcutaneous, intraperitoneal,intranasal, oral or other mucosal routes. Additional routes includeintracranial (for example intracisternal, or intraventricular),intraorbital, ophthalmic, intracapsular, intraspinal, and topicaladministration. The adjuvants and vaccine compositions of the inventioncan be administered in a suitable, nontoxic pharmaceutical carrier, orcan be formulated in microcapsules or a sustained release implant. Theimmunogenic compositions of the invention can be administered multipletimes, if desired, in order o sustain the desired cellular immuneresponse. The appropriate route, formulation, and immunization schedulecan be determined by one skilled in the art.

In the methods of the invention, in some instances the antigen and a NKTactivator/CD40 agonist conjugate may be administered separately orcombined in the same formulation. In some instances it may be useful toinclude several antigens. These compositions may be administeredseparately or in combination in any order that achieve the desiredsynergistic enhancement of cellular immunity. Typically, thesecompositions are administered within a short time of one another, i.e.within about several hours of one another, more preferably within abouta half hour. In some embodiments they may be co-administered withinabout 24-48 hours of one another.

In some instances, it may be beneficial to include a moiety in theadjuvant which facilitates affinity purification. Such moieties includerelatively small molecules that do not interfere with the function ofthe adjuvant combination. Alternatively, the tags may be removable bycleavage. Examples of such tags include poly-histidine tags,hemagglutinin tags, maltase binding protein, lectins, glutathione-Stransferase, avidin and the like. Other suitable affinity tags includeFLAG, green fluorescent protein (GFP), myc, and the like.

The subject adjuvant combinations can be administered with aphysiologically acceptable carrier such as physiological saline. Thecomposition may also include another carrier or excipient such asbuffers, such as citrate, phosphate, acetate, and bicarbonate, aminoacids, urea, alcohols, ascorbic acid, phospholipids, proteins such asserum albumin, ethylenediamine tetraacetic acid, sodium chloride orother salts, liposomes, mannitol, sorbitol, glycerol and the like. Theadjuvants of the invention can be formulated in various ways, accordingto the corresponding route of administration. For example, liquidformulations can be made for ingestion or injection, gels or procedurescan be made for ingestion, inhalation, or topical application. Methodsfor making such formulations are well known and can be found in forexample, “Remington's Pharmaceutical Sciences,” 18^(th) Ed., MackPublishing Company, Easton Pa.

The invention also embraces DNA based vaccines. These DNAs which mayencode a desired antigen and/or CD40 adjuvant may be administered asnaked DNAs, or may be comprised in an expression vector. Furthermore,the subject nucleic acid sequences may be introduced into a cell of agraft prior to transplantation of the graft. This DNA preferably will behumanized to facilitate expression in a human subject.

The subject adjuvant combinations may further include a “marker” or“reporter.” Examples of marker or reporter molecules include betalactamase, chloramphenicol acetyltransferase, adenosine deaminase,aminoglycoside phosphotransferase, dihydrofolate reductase, hygromycinB-phosphotransferase, thymidine kinase, lacZ, and xanthine guaninephosphoribosyltransferase et al.

Prokaryotic and eukaryotic cells that can be used to facilitateexpression of the subject adjuvants or antigens include by way ofexample microbia, plant and animal cells, e.g., prokaryotes such asEscherichia coli, Bacillus subtilis, and the like, insect cells such asSf21 cells, yeast cells such as Saccharomyces, Candida, Kluyveromyces,Schizzosaccharomyces, and Pichia, and mammalian cells such as COS,HEK293, CHO, BHK, NIH 3T3, HeLa, and the like. One skilled in the artcan readily select appropriate components for a particular expressionsystem, including expression vector, promoters, selectable markers, andthe like suitable for a desired cell or organism. The selection and useof various expression systems can be found for example in Ausubel etal., “Current Protocols in Molecular Biology, John Wiley and Sons, NewYork, N.Y. (1993); and Pouwels et al., Cloning Vectors: A LaboratoryManual”:, 1985 Suppl. (1987). Also provided are eukaryotic cells thatcontain and express the subject DNA constructs.

In the case of cell transplants, the cells can be administered either byan implantation procedure or with a catheter-mediated injectionprocedure through the blood vessel wall. In some cases, the cells may beadministered by release into the vasculature, from which the cellssubsequently are distributed by the blood stream and/or migrate into thesurrounding tissue.

The “NKT activators or agonists” useful in the invention include allactivators or agonists of NKT cells generally known in the art. Examplesof NKT activators r agonists include for example the ceramide and otherglycolipid compounds disclosed inn US20060211856 and US20060052316, bothof which published patent applications are incorporated by reference intheir entirety herein. For example this includes alpha-galactosylceramide, alpha-glucosyl ceramide, phosphatidylinisitol andglycosylphosphatidylinisitol and the various other glycolipid compoundsand derivatives disclosed in the cited patents as NKT activators andknown in the art. See also Parekh et al., Crit. Rev. Immunol.25)₃):183-213 (2005); and Parekh et al., J. Immunol. 173(1):3693-706(2006), both of which references are also incorporated by reference intheir entirety herein.

The “CD40 agonists” useful in the invention as noted preferably comprisean agonistic anti-CD40 antibody or fragment thereof that specificallybinds CD40, preferably murine or human CD40 or a CD40L protein,derivative, multimer such as a trimeric CD40L conjugate. As used herein,the term “antibody” is used in its broadest sense to include polyclonaland monoclonal antibodies, as well as antigen binding fragments thereof.This includes Fab, F(ab′)₂, Fd and Fv fragments.

In addition the term “antibody” includes naturally antibodies as well asnon-naturally occurring antibodies such as single chain antibodies,chimeric antibodies, bifunctional and humanized antibodies. Preferredfor use in the invention are chimeric, humanized and fully humanantibodies. Methods for synthesis of chimeric, humanized, CDR-grafted,single chain and bifunctional antibodies are well known to those skilledin the art. These antibodies may comprise human IgG1, IgG2, IgG3 or IgG4constant regions. In addition, antibodies specific to CD40 are widelyknown and available and can be made by immunization of a suitable hostwith a CD40 antigen, preferably human CD40.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.

EXAMPLES Synergy of CD40 Agonists with α-GalCer in InducingAntigen-Specific CD8 Responses

The following experiments reveal that that a NKTalpha-galactosylceramide (α-GalCer) synergizes with anti-CD40 antibodiesin expanding CD8 cells with lytic function, similar to what has beenpreviously observed with TLR agonists. Combined administration of a CD40agonist and α-GalCer induced high levels of cell-mediated immunity.

Example 1 Combined Administration of Anti-CD40 and α-GalCerSynergistically Enhances the Expansion of Antigen-Specific T Cells InVivo

This example relates to the experiment contained in FIG. 1. Therein micewere administered 500 ug Ovalbumin, 100 ug anti-CD40, 100 ug S-27609(TLR7 agonist), 4 ug α-GalCer, i.v. in the combinations noted in thefigure. Five days later, standard CTL assay with Siinfekl pulsed targetswas performed, as previously described. On day six after administrationof the reagents noted, the number of OVA-specific, CD8⁺ T cells wasdetermined by tetramer staining, as previously described as well as thenumber of antigen-specific CD8⁺ T cells producing IFNγ afterrestimulation in vitro.

The results in FIG. 1 surprisingly demonstrate that the administrationof a neoantigen (OVA) in combination with a CD40 agonist (anati-CD40)and a TLR agonist (S-27609) or anti-CD40 with α-GalCer induces highlevels of OVA-specific, CD8⁺ T cells (determined by tetramer stainingusing a OVA-H-2b tetramer) and recall responses of OVA-specific CD8⁺ Tcells to produce IFN-α.

Example 2 Quantifying Immune Responses as a Result of Immunization withCombined Anti-CD40 and α-GalCer (Based on Data in FIG. 1) andDemonstration of Synergy of CD40 Agonists with α-GalCer in InducingProtective Anti-Tumor Immunity

Because the administration of anti-CD40/α-GalCer/OVA oranti-CD40/609/OVA as compared to the use of the agents alone asmonotherapies is far more effective at enhancing the total number ofantigen-specific CD8⁺ T cells, combination therapy was tested as aneffective means to induce therapeutic immunity in a murine model ofmelanoma. Initial studies using anti-CD40 and TLR* or α-GalCerdemonstrated that the administration of this combination of activatorstogether with peptide or whole protein elicited extremely high (>5%tetramer⁺ T cells of the CD8⁺ T cells) frequencies of antigen-specificcytotoxic T cells. We then wanted to explore whether high frequencies of“self-reactive or tumor-reactive” T cells could be elicited to protectagainst a syngeneic tumor. Tyrosinase-related protein-2 (TRP-2) is aprotein involved in melanin synthesis and has been used as amelanoma-associated antigen for immunotherapy. TRP-2 is expressed innormal melanocytes and melanomas and has been identified as a humantumor antigen recognized by CTLs and subsequently identified as a tumorrejection antigen for B16 melanoma. CD8⁺ T cell peptide epitopes havebeen described both for human and mouse. To test if anti-self-reactiveCD8⁺ T cells could be induced using the CD40/TLR platform, mice whichwere given B16 melanoma (four days prior) were immunized i.p with 50 ugof a heterocyclic version of TRP-2 (ΔV peptide; TRP-2 (SVYDFFVWL),anti-CD40 and TLR7* or anti-CD40 and α-GalCer (and monotherapies ofeach).

The results in FIG. 2 show that the most effective therapeuticprotection was afforded by the combined use of anti-CD40/TLR/ΔV oranti-CD40/α-GalCer/ΔV, as judged by the number of lung metastases 21days after administration of the tumor. Some prophylactic protection isafforded by α-GalCer and ΔV. However, based on our experience with othertherapies, the use of anti-CD40/α-GalCer/ΔV will result in enhancedsurvival of those mice, as compared to mice receiving α-GalCer/ΔVtherapy.

The in vivo killing assay also suggests that combination therapy(OVA/anti-CD40/α-GalCer) is also more effective then any monotherapy(anti-CD40/OVA or α-GalCer/OVA or TLR/OVA) tested (see FIG. 2).

Example 3 CD40/TLR* Vaccination Therapeutically Intervenes in SystemicMelanoma

This example relates to the experiment contained in FIG. 3. In thisexperiment contained in FIG. 3, B6 mice (4/group) were immunized fourdays after the i.v. injection of 100K B16-F10 tumor cells. Particularly,mice were immunized with the combination of agents noted: Anti-CD40 (100ug/mouse, i.p.), S27609 (TLR7*) 100 ug), ΔV-peptide (100 ug), orα-GalCer (4 ug), i.v. Twenty days after administration of tumor (using aDay +4 intervention scheme) lungs were removed (see representativephoto; top in FIG. 3) and the number of lung metastases quantified byvisual counting (see bottom panel of FIG. 3).

Example 4 Anti-CD40 Antibody and α-GalCer Induces CD70 Expression on DCsIn Vivo

In the experiment contained in FIG. 4, mice were injected withcombinations of the following: 2 ug α-GalCer, 50 ug polyIC, and/or 50 uganti-CD40 i.p. Afterward, spleens were taken at 18-20 hours postinjection. The spleens were collagenase digested, stained for CD11c+ andanalyzed by CD8+ and CD8− (CD11b+) DCs.

The results in this Figure unexpectedly reveal that the expression ofCD70 is only induced on dendritic cells obtained from mice which wereadministered the combination of a CD40 agonist and α-GalCer.

The present inventors have previously reported that only when bothanti-CD40 and TLR agonists are administered in combination that there isan increase in CD70 expression on the surface of dendritic cells invivo. In addition, we have shown that expression of CD70 is essentialfor the subsequent expansion of antigen-specific CD8⁺ T cells.

Quite unexpectedly, similar results are observed with the administrationof the subject novel synergistic adjuvant combinations. Particularly,and similar to the synergy seen with TLR and CD40 agonists, upregulationof CD70 on DCs is also seen by the combined administration anti-CD40 andα-GalCer. As shown in FIG. 4, there is observed significantly increasedexpression of CD70 on CD8⁺ and CD8−, CD11c⁺ DCs only when anti-CD40 andTLR agonist (polyIC) or when anti-CD40 and α-GalCer are administered.

Example 5 α-GalCer/CD40 Immunization Elicits a Potent CD8+ T CellResponse that is STAT4, IFNγ and IFNab Independent but is STAT1 and TbetDependent

The Signal Transducers and Activator of Transcription (STAT) proteinsregulate many aspects of cell growth. STAT1 is involved in upregulatinggenes due to a signal by either type I or type II interferons. Inresponse to IFN-γ stimulation, STAT1 forms homodimers or heterodimerswith STAT3 that bind to the GAS (Interferon-Gamma Activated Sequence)promoter element; in response to either IFN-α or IFN-β stimulation,STAT1 forms a heterodimer with STAT2 that can bind the ISRE (InterferonStimulated Response Element) promoter element. The role of IL-12 incellular immunity is largely mediated by the STAT-4 transcriptionfactor. STAT-4 is essential for IL-12 activity. T-bet is a member of theT-box family of transcription factors that regulates lineage commitmentin CD4 (TH) lymphocytes. This is done part by activating IFN-γ. T-bet isrequired for control of IFN-production in CD4 and NK cells, but not inCD8 cells.

In the experiment contained in FIG. 5, mice (wtB6 or wtB6/129F1's andKO) were immunized I.V. with 50 ug ant-CD40 (FGK45), 2 ug α-GalCer, and200 ug of whole ovalbumin. Seven days later, the mice were sacrificedand PBLs and spleen cells isolated and stained with Kb/ova MHC tetramersto identify ova-specific CD8+ T cells as previously described (Sanchezet al. 2007 Journal of Immunology). The dot plots shown are gated on alllive, CD8+ B220− cells and the phenotype of the tetramer + and − cellsis shown with respect to the surface marker KLRG1. Numbers shownrepresent the percent tetramer+ out of total CD8+ T cells. Dot plotsshown are for PBL, but spleen reflects similar trends. This experimentindicates that α-GalCer/CD40 immunization elicits a potent CD8+ T cellresponse that is STAT4, IFNγ and IFNab independent but is STAT1 andT-bet independent.

Conclusions

Based at least on the results contained in the experiments in FIGS. 1-5,the combined administration of a CD40 agonist, together with antigen andan agonist that triggers NK-T cells (α-GalCer) induces immunity that isheightened over any of these agonists alone (elicits a synergisticeffect on immunity). The combination of CD40 agonist and α-GalCerresults in higher frequencies of antigen-specific CD8⁺ T cells, highernumbers of antigen-specific CD8⁺ T cells and enhance anti-tumor immunitywhen compared to any agonist alone with or without antigen.

Therefore, the combined use of a CD40 agonist and a NK-T cell activator,like α-GalCer, will serve as a potent vaccine platform in elicitingprotective cell-mediated immune responses to tumors and infectiousdisease.

The various references to journals, patents, and other publicationswhich are cited herein comprise the state of the art and areincorporated by reference as though fully set forth.

The invention is further defined by the claims which follow.

The invention claimed is:
 1. A method of promoting antitumor immunity ina subject in need thereof, consisting of the administration of acombination of adjuvants consisting of: (i) a human CD40 agonist, whichis selected from an agonistic CD40 antibody or an agonistic CD40antibody fragment or a multimeric CD40L polypeptide or multimeric CD40Lprotein fragment; (ii) alpha-galactosyl ceramide, and (iii) optionally atumor antigen, wherein the administration of said combination elicits asynergistic enhancement of CD70 expression on an immune cell relative toeither the CD40 agonist or the alpha galactosyl ceramide administered asa monotherapy.
 2. The method of claim 1, wherein the CD40 agonist is anagonistic CD40 antibody.
 3. The method of claim 2, wherein said antibodycontains a human constant domain.
 4. The method of claim 3, wherein theantibody is a human immunoglobulin.
 5. The method of claim 3, whereinthe human constant domain is an IgG1, IgG2, IgG3 or IgG4.
 6. The methodof claim 1, wherein the adjuvant combination includes a tumor antigen.7. The method of claim 1, wherein the cancer is a melanoma.
 8. Themethod of claim 7, wherein the melanoma is metastatic.
 9. The method ofclaim 1, wherein the immune cell is a dendritic cell.